Int J Stem Cells.  2025 Feb;18(1):49-58. 10.15283/ijsc24066.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells

• Kim J
• Park SH
• Sun W

• KMID: 2565196
• DOI: http://doi.org/10.15283/ijsc24066

Abstract

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Keyword

Valproic acid; Toxicity; Pluripotent stem cells; Organoids; Morphogenesis

Figure

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  Fig. 1 Toxicological assessment of valproic acid (VPA) exposure during anterior and posterior neural induction. (A) Representative bright field images. Scale bar=200 μm. VPA was exposed at concentrations ranging from 0 to 3 mM for 2 days during the induction of anterior neural stem cells (SB＋LDN) and posterior neural stem cells (SB＋CHIR). (B) Live cell number was measured by cell counter after single cell dissociation. N=3∼4. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; **p<0.01, ***p<0.001, ****p<0.0001. (C) Representative images after staining with acridine orange (AO)/propidium iodide (PI). Scale bar=100 μm. (D) Quantification of cell death by the percentage of PI-stained red fluorescence cells in AO-stained green fluorescence cells. N=9∼12. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; *p<0.05, ****p<0.0001.

• 

  Fig. 2 Analysis of the molecular mechanism underlying valproic acid (VPA)-induced cytotoxicity. (A) Representative images stained with phosphohistone H3 (PHH3), cell proliferation marker, and Hoechst33342 (HOE). Scale bar=100 μm. VPA (1 mM) was treated for 2 days during the anterior neural induction (SB＋LDN) and posterior neural induction (SB＋CHIR). The quantification was performed by the measurement of the percentage of cells stained with PHH3 among cells stained with HOE. N=14∼18. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; ****p<0.0001. (B) Representative images stained with cleaved-caspase 3, apoptotic marker, and HOE. Scale bar=100 μm. The quantification was performed by the measurement of the fluorescence intensity of cleaved-caspase 3 in the area where the colony was located. N=6∼7. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; ****p<0.0001. (C) Representative images of anteriorly guided neural stem cells stained with Cox4, mitochondrial marker, cytochrome c (CytC), and HOE after VPA treatment for 2 days. Scale bar=100 μm. (D) The changes of mRNA expression of BECN1, an autophagy marker, in anterior and posterior neural stem cells with treatment 1 mM VPA. N=4. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; ***p<0.001. (E) The alternation of mRNA expression of MLKL and RIPK1, which are necroptosis markers, and PARP1, a parthanatos marker in anterior and posterior neural stem cells with treatment 1 mM VPA. N=4. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; **p<0.01, ****p<0.0001.

• 

  Fig. 3 The effect of valproic acid (VPA) on anterior and posterior neural induction. (A) mRNA expression of SOX2, neural stem cell marker, and Brachyury T (Bra T), mesodermal marker. VPA was exposed at concentrations ranging from 0 to 1 mM for 2 days during the anterior (SB＋LDN) and posterior (SB＋CHIR) neural induction. N=4. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a two-way ANOVA with Tukey multiple comparisons; **p<0.01, ****p<0.0001. (B) Immunocytochemistry of SOX2 and Bra T. Scale bar=100 μm. VPA (1 mM) was exposed for 2 days during the anterior and posterior neural induction. The arrows show a profound expression of Bra T in the middle of the colonies. Scale bar=50 μm for an enlarged inset.

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  Fig. 4 The effect of valproic acid (VPA) pretreatment during the posterior neural induction step on neurulation-like tube morphogenesis in the neural organoid. (A) Experimental scheme of pretreatment of VPA during the posterior neural induction for 3 days, generation of neural organoids, and analysis of tube morphogenesis. (B) Time-lapse bright-field images of neural organoids in the VPA-pretreated group compared with control (Ctrl). Scale bar=200 μm. (C) The size of neural organoids was measured by the area of the organoids. Left: The neural organoid size at day 8 in the VPA-treated group compared by Ctrl. Right: Growth rate of neural organoids between day 5 to day 13. N=31∼32. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a one-way ANOVA with Tukey multiple comparisons; ****p<0.0001. (D) Quantification of neurulation-like tube morphogenesis. The color box indicated the cumulative proportion of the neural folding stage (left) and neural tube closure (right). On day 7, the neural organoids were cultured in differentiation media without basic fibroblast growth factor (bFGF) for 6 days and morphogenesis of three-dimensional structures was recorded in real-time. In each group, 31 to 32 neural organoids were used. (E) Representative 3D neural organoids images stained with ZO-1, tight junction protein, and Hoechst33342 (HOE). Rosettes were marked with red lines and shades in the right images. Scale bar=100 μm. (F) The percentage of rosette area per organoid area was quantified. N=4∼6. Data are presented as mean±SEM with individual data points. Statistical significance was tested with a one-way ANOVA with Tukey multiple comparisons; ****p<0.0001.

Reference

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